Shiga Toxin, Stx2e, Influences the Activity of Porcine Lymphocytes In Vitro.

Oedema disease (OD) in piglets is one of the most important pathologies, as it causes significant losses due to the high mortality because of the Shiga toxin family, which produces Escherichia coli (STEC) strains. The main toxin responsible for the characteristic pathologies in pigs is Shiga toxin 2 subtype e (Stx2e). Moreover, there is growing evidence that Stx's family of toxins also targets immune cells. Therefore, this study evaluated the effect of different concentrations of Stx2e on porcine immune cells. Porcine peripheral blood mononuclear cells were pre-incubated with Stx2e, at three different concentrations (final concentrations of 10, 500, and 5000 CD50/mL) and with a negative control group. Cells were then stimulated with polyclonal mitogens: concanavalin A, phytohemagglutinin, pokeweed mitogen, or lipopolysaccharides. Cell proliferation was assessed by BrdU (or EdU) incorporation into newly created DNA. The activation of the lymphocyte subsets was assessed by the detection of CD25, using flow cytometry. The toxin significantly decreased mitogen-driven proliferation activity, and the effect was partially dose-dependent, with a significant impact on both T and B populations. The percentage of CD25+ cells was slightly lower in the presence of Stx2e in all the defined T cell subpopulations (CD4+, CD8+, and γδTCR+)-in a dose-dependent manner. B cells seemed to be the most affected populations. The negative effects of different concentrations of Stx2e on the immune cells in this study may explain the negative impact of the subclinical course of OD.

Divergent Evolution of the Transcriptional Network Controlled by Snf1-Interacting Protein Sip4 in Budding Yeasts.

Cellular responses to starvation are of ancient origin since nutrient limitation has always been a common challenge to the stability of living systems. Hence, signaling molecules involved in sensing or transducing information about limiting metabolites are highly conserved, whereas transcription factors and the genes they regulate have diverged. In eukaryotes the AMP-activated protein kinase (AMPK) functions as a central regulator of cellular energy homeostasis. The yeast AMPK ortholog SNF1 controls the transcriptional network that counteracts carbon starvation conditions by regulating a set of transcription factors. Among those Cat8 and Sip4 have overlapping DNA-binding specificity for so-called carbon source responsive elements and induce target genes upon SNF1 activation. To analyze the evolution of the Cat8-Sip4 controlled transcriptional network we have compared the response to carbon limitation of Saccharomyces cerevisiae to that of Kluyveromyces lactis. In high glucose, S. cerevisiae displays tumor cell-like aerobic fermentation and repression of respiration (Crabtree-positive) while K. lactis has a respiratory-fermentative life-style, respiration being regulated by oxygen availability (Crabtree-negative), which is typical for many yeasts and for differentiated higher cells. We demonstrate divergent evolution of the Cat8-Sip4 network and present evidence that a role of Sip4 in controlling anabolic metabolism has been lost in the Saccharomyces lineage. We find that in K. lactis, but not in S. cerevisiae, the Sip4 protein plays an essential role in C2 carbon assimilation including induction of the glyoxylate cycle and the carnitine shuttle genes. Induction of KlSIP4 gene expression by KlCat8 is essential under these growth conditions and a primary function of KlCat8. Both KlCat8 and KlSip4 are involved in the regulation of lactose metabolism in K. lactis. In chromatin-immunoprecipitation experiments we demonstrate binding of both, KlSip4 and KlCat8, to selected CSREs and provide evidence that KlSip4 counteracts KlCat8-mediated transcription activation by competing for binding to some but not all CSREs. The finding that the hierarchical relationship of these transcription factors differs between K. lactis and S. cerevisiae and that the sets of target genes have diverged contributes to explaining the phenotypic differences in metabolic life-style.

Comparing Two Intestinal Porcine Epithelial Cell Lines (IPECs): Morphological Differentiation, Function and Metabolism.

The pig shows genetical and physiological resemblance to human, which predestines it as an experimental animal model especially for mucosal physiology. Therefore, the intestinal epithelial cell lines 1 and J2 (IPEC-1, IPEC-J2)--spontaneously immortalised cell lines from the porcine intestine--are important tools for studying intestinal function. A microarray (GeneChip Porcine Genome Array) was performed to compare the genome wide gene expression of IPECs. Different significantly up-regulated pathways were identified, like "lysosome", "pathways in cancer", "regulation of actin cytoskeleton" and "oxidative phosphorylation" in IPEC-J2 in comparison to IPEC-1. On the other hand, "spliceosome", "ribosome", "RNA-degradation" and "tight junction" are significantly down-regulated pathways in IPEC-J2 in comparison to IPEC-1. Examined pathways were followed up by functional analyses. ATP-, oxygen, glucose and lactate-measurement provide evidence for up-regulation of oxidative phosphorylation in IPEC-J2. These cells seem to be more active in their metabolism than IPEC-1 cells due to a significant higher ATP-content as well as a higher O2- and glucose-consumption. The down-regulated pathway "ribosome" was followed up by measurement of RNA- and protein content. In summary, IPEC-J2 is a morphologically and functionally more differentiated cell line in comparison to IPEC-1. In addition, IPEC-J2 cells are a preferential tool for in vitro studies with the focus on metabolism.

Systemic E. coli lipopolysaccharide but not deoxynivalenol results in transient leukopenia and diminished metabolic activity of peripheral blood mononuclear cells ex vivo.

The mycotoxin deoxynivalenol (DON) and lipopolysaccharides (LPS) are reported to act synergistically in the animal organism. Thus, we tested the hypothesis that systemic co-exposure of DON and LPS aggravates the impact of the individual toxin on leukocyte counts in vivo and peripheral blood mononuclear cells (PBMC) ex vivo. Growing barrows were fed a standard diet, equipped with permanent venous catheters and infused for 1 h with one of four treatments: control group with physiological saline (CON, n=8), mycotoxin group (DON, n=6) with 100 µg/kg body weight (BW) deoxynivalenol, endotoxin group (LPS, n=6) with 7.5 µg/kg BW Escherichia coli LPS, and co-exposed group (DON+LPS, n=6) with 100 µg/kg BW DON and 7.5 µg/kg BW LPS. Blood was collected 30 min prior to infusion and 10, 20, 30, 60, 360, 720 and 1440 min after start of infusion for total and differential leukocyte counts. PBMC were isolated from blood drawn at 3 and 24 h and subjected to an ex vivo 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay, either non-stimulated or stimulated with concanavalin A. LPS induced a transient significant leukopenia between 30 and 360 min, owing to a decrease in segmented neutrophils and lymphocytes (time×treatment: p<0.001). Metabolic activity of stimulated PBMC ex vivo was severely compromised in pigs 3 h after LPS exposure (<50% of control, p<0.001), but already regained 80% of its activity at 24 h, thus showing no difference between treatments. DON alone did not affect leukocytes in vivo or PBMC activity ex vivo and neither aggravated the effect of LPS.

Lipopolysaccharides (LPS) modulate the metabolism of deoxynivalenol (DON) in the pig.

Pigs might be exposed to lipopolysaccharides (LPS) and deoxynivalenol (DON) at the same time, and both toxins are thought to interactively affect the intestinal barrier, the innate immune system, and the xenobiotics metabolism. Hence, we aimed at examining the single and combined effects of both toxins on nutrient digestibility and DON metabolism. For this purpose, barrows (26 ± 4 kg) were fed restrictedly either a control diet (CON) or a diet contaminated with 3.1 mg DON/kg (DON) for 37 days. At day 37 of the experiment, pigs were infused intravenously for 60 min either with 100 µg DON/kg body weight (BW) (CON-DON), 7.5 µg LPS/kg BW (CON-LPS, DON-LPS) or a combination of both substances (CON-DON + LPS), or physiological saline (CON-CON, DON-CON). Blood samples were collected frequently until 3.25 h before the pigs were sacrificed for bile, liver, and kidney collection. The apparent digestibility of N-free extractives was significantly increased by 1 % when the DON-contaminated diet was fed. The total DON content in blood was significantly higher in endotoxemic pigs (34.8 ng/mL; CON-DON + LPS) when compared to the pigs infused with DON alone (18.8 ng/mL; CON-DON) while bile concentrations were not influenced by LPS. DON residue levels in liver and kidney closely reflected the treatment effects as described for blood. In contrast to DON infusion, the LPS challenge resulted in a significantly lower total DON concentration (13.2 vs. 7.5 ng/mL in groups DON-CON and DON-LPS, respectively) when the pigs were exposed to DON through the diet. The conjugation degree for DON in blood and bile was not influenced by treatments. In conclusion, endotoxemic pigs are characterized by higher DON residue levels in blood, liver, and kidney, probably by a compromised elimination.

Rapid interaction of Helicobacter pylori with microvilli of the polar human gastric epithelial cell line NCI-N87.

Infection with Helicobacter pylori results often in chronic gastritis, gastric ulcers or even gastric tumor development. Little is known about the initial interaction between gastric epithelial cells and H. pylori. The aim of the present study was to analyze the initial host contact to the bacteria. Monolayers of the human gastric epithelial cell line NCI-N87 grown on porous membranes were used and the apical side of the epithelium was exposed to the H. pylori wild-type strain P1 for 1 hr. Many epithelial cells were colonized by bacteria within the period of 60 min. Using scanning electron microscopy we detected that the bacteria were in close contact with the epithelia via microvilli. Further, transmission electron microscopy of the contact sites revealed no difference in the morphology of the microvilli in comparison to those not attached to the bacteria. The present study demonstrates the importance of microvilli on apical epithelial cells during the initial contact of the host by colonizing H. pylori.

The Fusarium toxin deoxynivalenol (DON) modulates the LPS induced acute phase reaction in pigs.

The systemic effects of the Fusarium toxin deoxynivalenol (DON) and of bacterial lipopolysaccharides (LPS) were studied in male castrated pigs (40.4 ± 3.7 kg) infused intravenously with either DON or LPS alone (100 µg DON/kg/h, 7.5 µg/LPS/kg/h), or together (100 µg DON plus 7.5 µg/LPS/kg/h). The Control group received a saline infusion (n=6/treatment, 24h observation period). An additional DON infusion did not exacerbate the clinical signs observed in LPS-infused pigs. For example, rectal temperature climaxed after 4h (40.4 ± 0.2°C) and 5h (40.1 ± 0.3°C), in the LPS and LPS+DON group, respectively. Saline and DON alone did not induce an acute phase reaction as indicated by unaltered plasma levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) while LPS caused a significant rise of both cytokines. TNF-alpha plasma peak concentrations were significantly higher in the LPS compared to the DON+LPS group (94.3 ± 17.2 ng/mL vs. 79.2 ± 15.7 ng/mL) while IL-6 climaxed earlier in the latter group (3h p.i. vs. 2h p.i.). From the tested clinical-chemical plasma characteristics the total bilirubin concentration and the ASAT activity were strongly elevated by the LPS infusion and additionally increased and decreased by DON, respectively. In conclusion, the LPS-induced effects were only marginally modified by DON.

A chronic oral exposure of pigs with deoxynivalenol partially prevents the acute effects of lipopolysaccharides on hepatic histopathology and blood clinical chemistry.

Lipopolysaccharides (LPS), a cell wall component of gram-negative bacteria, and deoxynivalenol (DON), a prevalent Fusarium-derived contaminant of cereal grains, are each reported to have detrimental effects on the liver. A potentiating toxic effect of the combined exposure was reported previously in a mouse model and hepatocytes in vitro, but not in swine as the most DON-susceptible species. Thus, pigs were fed either a control diet (CON) or a Fusarium contaminated diet (DON, 3.1mg DON/kg diet) for 37 days. At day 37 control pigs were infused for 1h either with physiological saline (CON_CON), 100µg/kg BW DON (CON_DON), 7.5µg/kg BW LPS (CON_LPS), or both toxins (CON_DON/LPS) and Fusarium-pigs with saline (DON_CON) or 7.5µg/kg BW LPS (DON_LPS). Blood samples were taken before and after infusion (-30, +30, +60, +120, and +180min) for clinical blood chemistry. Pigs were sacrificed at +195min and liver histopathology was performed. LPS resulted in higher relative liver weight (p<0.05), portal, periportal and acinar inflammation (p<0.05), haemorrhage (p<0.01) and pathological bilirubin levels (CON_CON 1.0µmol/L vs. CON_LPS 5.4µmol/L, CON_DON/LPS 8.3µmol/L; p<0.001). DON feeding alleviated effects of LPS infusion on histopathology and blood chemistry to control levels, whereas DON infusion alone had no impact.

The plasma clearance of the Fusarium toxin deoxynivalenol (DON) is decreased in endotoxemic pigs.

The plasma elimination kinetics of the Fusarium toxin deoxynivalenol (DON) was investigated in male castrated pigs (40.4±3.7 kg) when infused intravenously either alone (100 µg/kg/h, n=6) or together with lipopolysaccharides (LPS, 7.5 µg/kg/h, n=6). The maximum DON concentration after one hour of infusion was significantly higher by 61% in the DON+LPS Group compared to pigs infused with DON alone. The area under the plasma DON concentration vs. time curve of the DON+LPS Group was approximately twice as high as that of the DON Group after 24h while the initial (0.63 vs. 0.6 h) and terminal half-lifes (2.97 vs. 2.30 h) remained uninfluenced. The apparent volume of distribution and the plasma clearance were significantly lower for the DON+LPS Group compared to the DON Group (2.14 vs. 1.45 L/kg and 11.9 vs. 5.87 mL/kg/min). Glucuronidated DON seemed to persist longer in the DON+LPS Group. In conclusion, clearance of DON was decreased during an LPS induced acute phase reaction in pigs. Whether the higher plasma DON concentrations in endotoxemic pigs are due to a hemodynamically associated longer persistence of the DON glucuronide or because of an altered glucuronidation activity needs to be examined further.

Gene regulation of intestinal porcine epithelial cells IPEC-J2 is dependent on the site of deoxynivalenol toxicological action.

The intestinal epithelial cell layer represents the border between the luminal and systemic side of the gut. The decision between absorption and exclusion of substances is the quintessential function of the gut and varies along the gut axis. Consequently, potentially toxic substances may reach the basolateral domain of the epithelial cell layer via blood stream. The mycotoxin deoxynivalenol (DON) is a Fusarium derived secondary metabolite known to enter the blood stream and displaying a striking toxicity on the basolateral side of polarised epithelial cell layers in vitro. Here we analysed potential mechanisms of apical and basolateral DON toxicity reflected in the gene expression. We used the jejunum-derived, polarised intestinal porcine epithelial cell line IPEC-J2 as an in vitro cell culture model. Luminal and systemic DON challenge of the epithelial cell layer was mimicked by a DON application from the apical or basolateral compartment of membrane inserts for 72 h. We compared the genome-wide gene expression of untreated and DON-treated IPEC-J2 cells with the GeneChip® Porcine Genome Array of Affymetrix. Low basolateral DON (200 ng/mL) application triggered 10 times more gene transcripts in comparison to the corresponding apical application (2539 versus 267) despite the intactness of the challenged cell layer as measured by transepithelial electrical resistance. Analysis of the regulated genes by bioinformatic resource DAVID identified several groups of biochemical pathways modulated by concentration and orientation of DON application. Selected genes representing pathways of the cellular metabolism, information processing and structural design were analysed in detail by quantitative PCR. Our findings clearly show that apical and basolateral challenge of epithelial cell layers trigger different gene response profiles paralleled with a higher susceptibility towards basolateral challenge. The evaluation of toxicological potentials of mycotoxins should take this difference in gene regulation dependent on route of application into account.

Vulnerability of polarised intestinal porcine epithelial cells to mycotoxin deoxynivalenol depends on the route of application.

BACKGROUND AND AIMS: Deoxynivalenol (DON) is a Fusarium derived mycotoxin, often occurring on cereals used for human and animal nutrition. The intestine, as prominent barrier for nutritional toxins, has to handle the mycotoxin from the mucosa protected luminal side (apical exposure), as well as already absorbed toxin, reaching the cells from basolateral side via the blood stream. In the present study, the impact of the direction of DON exposure on epithelial cell behaviour and intestinal barrier integrity was elucidated. METHODS: A non-transformed intestinal porcine epithelial cell line (IPEC-J2), cultured in membrane inserts, serving as a polarised in vitro model to determine the effects of deoxynivalenol (DON) on cellular viability and tight junction integrity. RESULTS: Application of DON in concentrations up to 4000 ng/mL for 24, 48 and 72 hours on the basolateral side of membrane cultured polarised IPEC-J2 cells resulted in a breakdown of the integrity of cell connections measured by transepithelial electrical resistance (TEER), as well as a reduced expression of the tight junction proteins ZO-1 and claudin 3. Epithelial cell number decreased and nuclei size was enlarged after 72 h incubation of 4000 ng/mL DON from basolateral. Although necrosis or caspase 3 mediated apoptosis was not detectable after basolateral DON application, cell cycle analysis revealed a significant increase in DNA fragmentation, decrease in G0/G1 phase and slight increase in G2/M phase after 72 hours incubation with DON 2000 ng/mL. CONCLUSIONS: Severity of impact of the mycotoxin deoxynivalenol on the intestinal epithelial barrier is dependent on route of application. The epithelium appears to be rather resistant towards apical (luminal) DON application whereas the same toxin dose from basolateral severely undermines barrier integrity.

Mycotoxin deoxynivalenol (DON) mediates biphasic cellular response in intestinal porcine epithelial cell lines IPEC-1 and IPEC-J2.

The Fusarium derived mycotoxin deoxynivalenol (DON) is frequently found in cereals used for human and animal nutrition. We studied effects of DON in non-transformed, non-carcinoma, polarized epithelial cells of porcine small intestinal origin (IPEC-1 and IPEC-J2) in a low (200 ng/mL) and a high (2000 ng/mL) concentration. Application of high DON concentrations showed significant toxic effects as indicated by a reduction in cell number, in cellular reduction capacity measured by MTT assay, reduced uptake of neutral red (NR) and a decrease in cell proliferation. High dose toxicity was accompanied by disintegration of tight junction protein ZO-1 and increase of cell cycle phase G2/M. Activation of caspase 3 was found as an early event in the high DON concentration with an initial maximum after 6-8 h. In contrast, application of 200 ng/mL DON exhibited a response pattern distinct from the high dose DON toxicity. The cell cycle, ZO-1 expression and distribution as well as caspase 3 activation were not changed. BrdU incorporation was significantly increased after 72 h incubation with 200 ng/mL DON and NR uptake was only transiently reduced after 24 h. Low dose effects of DON on intestinal epithelial cells were triggered by mechanisms different from those responsible for the high dose toxicity.